Antitumor efficacy of IPI-504, a selective heat shock protein 90 inhibitor against human epidermal growth factor receptor 2-positive human xenograft models as a single agent and in combination with trastuzumab or lapatinib.

IPI-504 is a novel, highly soluble small-molecule inhibitor of heat shock protein 90 (Hsp90), a protein chaperone essential for regulating homeostasis of oncoproteins and cell signaling proteins. Human epidermal growth factor receptor 2 (HER2; ErbB2) oncoprotein, expressed in a subset of metastatic breast cancers, is a Hsp90 client protein. In this study, we investigated the antitumor activity and the mechanism of action of IPI-504 in HER2(+), trastuzumab-sensitive and trastuzumab-refractory cell lines in vitro and in vivo. IPI-504 exhibited potent antiproliferative activities (range of IC(50), 10-40 nmol/L) against several tumor cell lines examined, whereby mechanism of action was mediated through HER2 and Akt degradation. Both intravenous and oral administration of IPI-504 assessed in multiple schedules showed potent tumor growth inhibition in vivo with corresponding degradation of HER2. The tolerability and efficacy of IPI-504 combined with either trastuzumab or lapatinib were also investigated in HER2(+) tumor xenograft models. Combination of IPI-504 with trastuzumab significantly enhanced tumor growth delay and induced greater responses when compared with either agent alone. Although, as expected, trastuzumab alone did not exhibit any significant antitumor activity in the trastuzumab-resistant JIMT-1 model, IPI-504 administered in combination with trastuzumab yielded greater antitumor efficacy than either agent alone. Finally, combination of IPI-504 and lapatinib was well tolerated up to 50 mg/kg IPI-504 and 100 mg/kg lapatinib and resulted in significant delay in tumor growth, including partial and complete tumor responses. These lines of evidence support the development of IPI-504 in HER2-positive breast cancers as a single agent and in combination with either trastuzumab or lapatinib

Antibody-dependent cell-mediated cytotoxicity effector-enhanced EphA2 agonist monoclonal antibody demonstrates potent activity against human tumors.

EphA2 is a receptor tyrosine kinase that has been shown to be overexpressed in a variety of human tumor types. Previous studies demonstrated that agonist monoclonal antibodies targeting EphA2 induced the internalization and degradation of the receptor, thereby abolishing its oncogenic effects. In this study, the in vitro and in vivo antibody-dependent cell-mediated cytotoxicity (ADCC) activity of EphA2 effector-enhanced agonist monoclonal antibodies was evaluated. With tumor cell lines and healthy human peripheral blood monocytes, the EphA2 antibodies demonstrated approximately 80% tumor cell killing. In a dose-dependent manner, natural killer (NK) cells were required for the in vitro ADCC activity and became activated as demonstrated by the induction of cell surface expression of CD107a. To assess the role of NK cells on antitumor efficacy in vivo, the EphA2 antibodies were evaluated in xenograft models in severe compromised immunodeficient (SCID) mice (which have functional NK cells and monocytes) and SCID nonobese diabetic (NOD) mice (which largely lack functional NK cells and monocytes). Dosing of EphA2 antibody in the SCID murine tumor model resulted in a 6.2-fold reduction in tumor volume, whereas the SCID/nonobese diabetic model showed a 1.6-fold reduction over the isotype controls. Together, these results demonstrate that the anti-EphA2 monoclonal antibodies may function through at least two mechanisms of action: EphA2 receptor activation and ADCC-mediated activity. These novel EphA2 monoclonal antibodies provide additional means by which host effector mechanisms can be activated for selective destruction of EphA2-expressing tumor cells.

Amifostine (ETHYOL) protects rats from mucositis resulting from fractionated or hyperfractionated radiation exposure.

PURPOSE: The cytoprotective drug amifostine (Ethyol) protects rats from oral mucositis resulting from a single dose of gamma-irradiation. We expanded earlier studies to determine whether multiple doses of amifostine protect against fractionated or hyperfractionated radiation and whether the active metabolite of amifostine (WR-1065) accumulates in tissues upon repeated administration. METHODS AND MATERIALS: Rats received amifostine daily for 5 days in conjunction with a 1-week fractionated radiation schedule and were evaluated for oral mucositis. Rats also received amifostine before the am or pm exposure or b.i.d. in conjunction with hyperfractionated radiation. To determine the pharmacokinetics of WR-1065 after repeated dosing, amifostine was given 5 days a week for 1 or 3 weeks, and rat tissue and plasma were collected at intervals during and after treatment and analyzed for WR-1065. RESULTS: Amifostine protected rats from mucositis resulting from fractionated or hyperfractionated radiation. When the number of days of amifostine administration was reduced, protection was diminished. A dose of 100 mg/kg given in the morning or 2 doses at 50 mg/kg provided the best protection against hyperfractionated radiation. WR-1065 did not accumulate in tissues or tumor upon repeated administration. CONCLUSIONS: Amifostine prevented radiation-induced mucositis in a rat model; protection was dose and schedule dependent.

Tissue levels of WR-1065, the active metabolite of amifostine (Ethyol), are equivalent following intravenous or subcutaneous administration in cynomolgus monkeys.

Amifostine (Ethyol) is a cytoprotective drug approved for the reduction of xerostomia in head and neck cancer when administered to patients receiving postoperative radiation therapy. Although amifostine is approved for intravenous infusion, the off-label subcutaneous route of administration has become more prevalent. Although human patient data indicate higher plasma bioavailability of the active metabolite (WR-1065) following intravenous compared to subcutaneous administration, there are no corresponding data showing human tissue levels of WR-1065 following either route of administration due to the difficulty in obtaining human specimens. In our study we compared plasma and tissue pharmacokinetics of WR-1065 in primates following both routes of administration. Monkeys received amifostine at a dose of 260 mg/m2 either intravenously or subcutaneously. Plasma samples were analyzed for total WR-1065 by reverse-phase high-pressure liquid chromatography (HPLC) and fluorescence detection up to 4 h after amifostine administration. Tissues were analyzed for free WR-1065 by reverse-phase HPLC and electrochemical detection 30 and 60 min after administration. Following intravenous administration, plasma WR-1065 levels peaked rapidly and showed a bi-exponential decline, while following subcutaneous administration WR-1065 levels rose slowly and declined exponentially. The relative plasma bioavailability of WR-1065 given subcutaneously was lower at 30 and 60 min. Interestingly, after 30 min, tissues showed equal or slightly greater concentrations of WR-1065 following subcutaneous administration. Levels following 60 min were comparable following both routes. The plasma bioavailability studies performed in primates confirm human plasma data. Expanding the study to evaluate primate tissue levels of WR-1065 revealed that despite lower plasma bioavailability following subcutaneous administration, tissue levels of the active metabolite were surprisingly greater than or equal to those measured in animals that received the drug intravenously. These studies strengthen the argument for subcutaneous administration of amifostine in radiation oncology.

Subcutaneous administration of amifostine (ethyol) is equivalent to intravenous administration in a rat mucositis model.

PURPOSE: Amifostine (Ethyol) is currently approved for intravenous (IV) administration to prevent xerostomia in patients receiving radiotherapy for head-and-neck cancer. Recently, subcutaneous (SC) administration has been explored as an alternative route. To determine whether SC administration was equivalent to IV administration, we used models to follow pharmacokinetics and oral mucosal protection in rats. METHODS: Amifostine was administered to rats at doses of 200, 100, or 50 mg/kg (1300, 650, or 325 mg/m(2)) IV or SC at various times before radiation at 15.3 Gy (protection studies) or harvest of blood and tissues for analysis by HPLC (pharmacokinetic studies). RESULTS: Amifostine administered IV or SC 1 h before radiation protected rats from mucositis, but the protective effect was more prolonged when amifostine was administered SC. Tissue levels of the active metabolite (WR-1065) were equivalent after SC administration. The correlation between tissue levels of WR-1065 and protection was strong, but that between blood levels of WR-1065 and protection was only weak. CONCLUSION: These data demonstrate that, in a rat model, SC administration of amifostine was at least as effective as that by IV.

Effects of dose and schedule on the efficacy of ethyol: preclinical studies.

The chemo- and radioprotectant drug amifostine (Ethyol; MedImmune, Inc, Gaithersburg, MD) is approved for intravenous (IV) administration; however, the subcutaneous (SC) route is being explored as a practical alternative. We have previously reported equivalence between IV and SC administration using a rat model of radioprotection and active metabolite (WR-1065) tissue pharmacokinetics. To examine the more clinically relevant fractionated and hyperfractionated radiation schedules and the effects of variations in the time of amifostine administration, we expanded these studies to include radioprotection and pharmacokinetic studies of WR-1065 using multiple dosing. To measure radioprotection using a fractionated radioprotection model, rats were given amifostine over a 1-week period at various doses (25 mg/kg, 50 mg/kg, 100 mg/kg; or 162.5 mg/m(2), 325 mg/m(2), 650 mg/m(2), respectively) IV or SC daily 30 minutes before exposure to 7.5 Gy/dose. Rats were fully protected from mucositis at the highest amifostine dose, with protection diminishing as the amifostine was decreased. Equivalent protection was observed whether the drug was given IV or SC. When the number of days of amifostine administration was reduced, protection was diminished. Amifostine also protected against radiation delivered using a 1-week hyperfractionated schedule (4.5 Gy/exposure twice daily), with optimal protection occurring when the drug was administered bid 30 minutes before each exposure (50 mg/kg) or every day before the morning exposure (100 mg/kg). The need for daily dosing to achieve optimal radioprotection was consistent with the tissue pharmacokinetics of the active metabolite. We found that WR-1065 did not accumulate in tissues or in SC-implanted tumors when amifostine was administered daily for 3 weeks. In addition, tissue and tumor levels of WR-1065 declined to baseline 24 hours after each amifostine dose. In a monkey pharmacokinetic model, plasma levels of WR-1065 (characterized by a pronounced spike of WR-1065 immediately after IV administration that was absent when the drug was given SC) were similar to those of humans; however, levels of WR-1065 in the tissues were higher 30 minutes following SC administration and were equivalent 60 minutes following IV or SC administration. These results suggest that maximum tissue levels and protection occur when amifostine is given 30 to 60 minutes before radiation exposure, that treatment breaks reduce the radioprotection by amifostine, and that protection from hyperfractionated radiation is dependent on amifostine dose and schedule.

Preclinical modeling of improved amifostine (Ethyol) use in radiation therapy.

Amifostine (Ethyol) has been evaluated clinically as a radioprotective agent for the prevention of xerostomia and mucositis for patients receiving radiotherapy (RT). Currently, amifostine is approved for the prevention of xerostomia in head and neck cancer patients receiving RT when administered intravenously (IV) before RT. For the clinician, there would be several advantages to administering the drug subcutaneously and to being able to show its protective effects on mucositis. The authors have developed a rat RT model to examine the protective effects of amifostine after IV and subcutaneous (SC) administration in a mucositis model. Rats (5 per group) were given 200 mg/kg (human dose equivalent of approximately 1,300 mg/m(2)) of amifostine either IV or SC, and their head and neck regions were exposed to 15.3 Gy of gamma radiation 0.5, 2, 4, and 8 hours after amifostine administration. For 10 days after treatment, the oral cavities of the rats were examined for signs of mucositis. Mucosal erythema and mucosal edema were scored according to 0 through 5 and 0 through 2 scales, respectively, with the scores added to indicate overall mucositis. The average mucositis score for the untreated animals was 3.5. Rats were protected from mucositis up to 4 hours when given amifostine either IV or SC. Rats that received amifostine SC, but not IV, were protected from mucositis 8 hours after administration. Preliminary pharmacokinetic data have revealed slightly higher active metabolite (WR-1065) levels in the parotid gland and small intestine in the rats given amifostine SC compared with IV and equivalent levels in the plasma and kidney. The data showed that SC administration of amifostine gave radioprotection comparable to IV administration up to 4 hours before RT and may be more effective than IV administration at longer pretreatment intervals.

Preclinical studies on the radioprotective efficacy and pharmacokinetics of subcutaneously administered amifostine.

The radioprotective effects and pharmacokinetics of subcutaneously (SC) administered amifostine have been investigated in animal studies. Studies in rats using a single dose of amifostine showed that SC administration gave protection from radiation-induced mucositis that is at least equivalent to that achieved by intravenous administration of the drug. These studies also indicate that tissue levels of the active metabolite WR-1065 correlated better with the radioprotective effects of amifostine than do plasma WR-1065 levels. Multiple-dose studies in rats show radioprotective effects equal to or greater than those obtained with intravenous dosing in the setting of fractionated irradiation. In addition, there is no evidence of drug accumulation in either normal or tumor tissue, with tumor WR-1065 levels peaking just above the limits of quantitation during treatment. Preliminary data from studies of SC amifostine in monkeys indicate a plasma pharmacokinetic profile similar to that reported earlier in humans. Tissue WR-1065 levels were higher at 30 minutes after SC dosing than they were after intravenous dosing and were comparable for the two routes at 60 minutes.